Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach

Mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs), a valuable tool for in vitro disease modeling and regenerative medicine. These applications demand for iPSCs devoid of reprogramming factor transgenes, but current procedures for the derivation of transgene-free iPSCs are inefficient and cumbersome. Here, we describe a new approach for the simple derivation of transgene-free iPSCs by the sequential use of two DNA recombinases, C31 Integrase and Cre, to control the genomic insertion and excision of a single, non-viral reprogramming vector. We show that such transgene-free iPSCs exhibit gene expression profiles and pluripotent developmental potential comparable to genuine, blastocyst-derived embryonic stem cells. As shown by a reporter iPSC line for the differentiation into midbrain dopaminergic neurons, the dual recombinase approach offers a simple and efficient way to derive transgene-free iPSCs for studying disease mechanisms and cell replacement therapies.     

Adenylate-Rich Sequences in Sendai Virus Transcripts from Infected Cells

Virus-specific complementary ribonucleic acid (RNA) from cells infected with Sendai virus was isolated by a procedure involving hybridization with virion RNA and isopycnic centrifugation of the RNA hybrids. The complementary RNA contained adenylate-rich sequences which sedimented at about 4S.     

XJ-160病毒复制子型表达载体的构建

XJ-160病毒是我国首次分离的辛德毕斯病毒,全基因组测序已经完成.本文利用该病毒全基因序列首先构建了全基因组cDNA克隆质粒,在此基础上,利用基因重组技术将病毒结构基因序列替换为含有多个单酶切位点的序列,得到复制子表达载体质粒pRepxj160.为验证载体的功能,将报告基因绿色荧光蛋白(EGFP)和β-半乳糖苷酶基因(lacZ)分别插入到载体的多克隆位点,得到两个表达质粒;经体外转录获得的转录体RNA转染BHK-21细胞后14h,可检测到报告基因的表达.结果表明我们构建的XJ-160病毒复制子型表达载体具有自主复制功能,可以表达异源基因.本研究为进一步开发具有我国自主知识产权的甲病毒载体奠定了基础.     

Infective Substructures of Sendai Virus from Infected Ehrlich Ascites Tumor Cells

Increase of infectivity for embryonated eggs was observed in Ehrlich ascites tumor cells after intraperitoneal inoculation of Sendai virus into tumor-bearing mice. Virus-induced actinomycin-resistant ribonucleic acid consisting of 14S, 18S, 22S, 35S, and 48S was synthesized, and S antigen was produced in infected cells. The infectivity was suggested to be due to viral ribonucleoprotein for the following reasons: (i) the infectivity was unaffected by V antiserum but was abolished by whole hyperimmune serum, (ii) the infectivity was resistant to ribonuclease, (iii) virus particles were found neither in cells nor on red blood cell stroma treated with cellular extracts, (iv) structures similar to Sendai virus ribonucleoprotein with a maximal length of 10,500 A were observed in cellular extracts.     

XJ—160病毒复制子型表达载体的构建

D-160病毒是我国首次分离的辛德毕斯病毒,全基因组测序已经完成。本文利用该病毒全基因序列首先构建了全基因组cDNA克隆质粒,在此基础上,利用基因重组技术将病毒结构基因序列替换为含有多个单酶切位点的序列,得到复制子表达载体质粒pRepxjl60。为验证载体的功能,将报告基因绿色荧光蛋白(EGFP)和β-半乳糖苷酶基因(lacZ)分别插入到载体的多克隆位点,得到两个表达质粒;经体外转录获得的转录体RNA转染BHK-21细胞后14h,可检测到报告基因的表达。结果表明我们构建的XJ-160病毒复制子型表达载体具有自主复制功能,可以表达异源基因。本研究为进一步开发具有我国自主知识产权的甲病毒载体奠定了基础。     

In Vitro Reconstruction of Neuronal Networks Derived from Human iPS Cells Using Microfabricated Devices

Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions.     

Fate of Sendai Virus Ribonucleoprotein in Virus-infected Cells

The cytoplasmic extracts of Ehrlich ascites tumor cells infected with (32)PO(4) and (3)H-leucine-labeled Sendai virus have been examined during the course of infection with respect to sedimentation behavior and buoyant densities of input virus radioactivity. It was found that (32)P and (3)H radioactivities were coincident, and, at 30 min after infection, the bulk of radioactivity was recovered in the polysome region of a sucrose gradient in the position of Sendai virus ribonucleoprotein (210S). The heterogeneity of radioactivity profiles appeared at 1 hr after infection and increased during 6 hr of incubation. The buoyant densities of input virus components were determined by banding in CsCl gradient. Here again the bulk of coincident (32)P and (3)H radioactivity at 30 min after infection banded at the same density as Sendai virus ribonucleoprotein (1.31 g/cm(3).) This component disappeared at 3 hr after infection, and (32)P and (3)H radioactivities were now found in components banded at densities 1.38, 1.41, 1.45, 1.49, and 1.55 g/cm(3). The results presented are consistent with the idea that virus ribonucleoprotein is retained in the cytoplasm of infected cells during at least 6 hr of incubation, being partly deproteinized in the course of infection. The nature of components which banded at rho = 1.41, 1.45, 1.49, and 1.55 as complexes of partly deproteinized ribonucleoprotein with ribosomes will be described in a separate paper.     

Circulating Angiogenic Cells can be Derived from Cryopreserved Peripheral Blood Mononuclear Cells

Background

Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs) for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs).

Methods

PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw) or 28 (late thaw) days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential.

Results

The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34⁺VEGFR2⁺CD133⁺ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle.

Conclusion

Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34⁺VEGFR2⁺CD133⁺ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.     

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.     

MicroRNA Expression Profiles of Human iPS Cells,Retinal Pigment Epithelium Derived From iPS,and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.     

一种基于塞姆利基森林病毒复制子的新型复制子载体

RNA复制子是能自主复制的病毒RNA。基于RNA复制子的表达载体和基因疫苗比常规真核表达载体和DNA疫苗具有更大的优越性。以塞姆利基森林病毒RNA复制子衍生的真核表达载体pSFV1为骨架 ,插入CMV立即早期启动子和SV40晚期Poly(A)信号 ,构建了一种完全基于DNA的复制型表达载体pSFV1CS ,将增强型绿色荧光蛋白基因EGFP插入其中 ,构建了重组质粒pSFV1CS EGFP ,通过转染 2 93T细胞 ,证实外源基因能在其中高效表达。该载体可用于表达真核蛋白和构建复制子载体疫苗。     

Enhancement of 32P Incorporation into Phospholipids in Cultured Cells by Sendai Virus of Parainfluenza Type 1

Sendai virus infection induced enhancement of ³²P incorporation into phospholipids in chick embryo, monkey kidney and bovine kidney cells, as previously observed in chorioallantoic membranes of chick embryos. These findings indicate that phospholipid synthesis is enhanced upon Sendai virus infection. Ultraviolet irradiation abolished the ability of the virus to induce the enhanced synthesis of phospholipids, a fact suggesting that the phenomenon depends upon infectivity of the virus. Gamma irradiation of host cells little affected the enhanced ³²P labeling of cellular phospholipids, suggesting that the function of host cell DNA may not be directly involved in the phenomenon.     

Cell Type Mediated Resistance of Vesicular Stomatitis Virus and Sendai Virus to Ribavirin

Ribavirin (RBV) is a synthetic nucleoside analog with broad spectrum antiviral activity. Although RBV is approved for the treatment of hepatitis C virus, respiratory syncytial virus, and Lassa fever virus infections, its mechanism of action and therapeutic efficacy remains highly controversial. Recent reports show that the development of cell-based resistance after continuous RBV treatment via decreased RBV uptake can greatly limit its efficacy. Here, we examined whether certain cell types are naturally resistant to RBV even without prior drug exposure. Seven different cell lines from various host species were compared for RBV antiviral activity against two nonsegmented negative-strand RNA viruses, vesicular stomatitis virus (VSV, a rhabdovirus) and Sendai virus (SeV, a paramyxovirus). Our results show striking differences between cell types in their response to RBV, ranging from virtually no antiviral effect to very effective inhibition of viral replication. Despite differences in viral replication kinetics for VSV and SeV in the seven cell lines, the observed pattern of RBV resistance was very similar for both viruses, suggesting that cellular rather than viral determinants play a major role in this resistance. While none of the tested cell lines was defective in RBV uptake, dramatic variations were observed in the long-term accumulation of RBV in different cell types, and it correlated with the antiviral efficacy of RBV. While addition of guanosine neutralized RBV only in cells already highly resistant to RBV, actinomycin D almost completely reversed the RBV effect (but not uptake) in all cell lines. Together, our data suggest that RBV may inhibit the same virus via different mechanisms in different cell types depending on the intracellular RBV metabolism. Our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro.     

Cytopathogenesis of Sendai Virus in Well-Differentiated Primary Pediatric Bronchial Epithelial Cells

Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector, as it is considered nonpathogenic in humans. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures are comprised of polarized pseudostratified multilayered epithelium containing ciliated, goblet, and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently and progressively and was restricted to ciliated and nonciliated cells, not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures postinfection. This manifested itself by ciliostasis, cell sloughing, apoptosis, and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of proinflammatory chemokines, including IP-10, RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), interleukin 6 (IL-6), and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest that caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.Sendai virus (SeV) is a nonsegmented negative-strand RNA virus of the Paramyxoviridae family. Recombinant SeV (rSeV) has been extensively studied as a vector for vaccines, cancer immunotherapy, and gene therapy (14, 22, 34, 41, 43). SeV is virulent in rodents, but despite extensive antigenic and genetic similarity to human parainfluenza virus type 1 (hPIV1), it is not known to cause disease in humans (33). Interest in rSeV as a vector is exemplified by the fact that (i) its genome can easily be manipulated to stably express heterologous genes (9), (ii) it does not undergo homologous recombination, (iii) cell transduction is independent of the cell cycle, (iv) there is no DNA phase during replication and hence no possibility of cell transformation, and (v) its cell or tissue tropisms and replication competency may be modulated by reverse genetics and appropriate rescue systems (5, 8).Much of the research on rSeV as a vector involves monolayer cells and animal models and employs both replication-competent and transmission-incompetent viruses. In view of its respiratory tract tropism, particular attention was paid to its use as a gene therapy vector for lung diseases such as cystic fibrosis (CF) (2, 13, 14, 43). Indeed, early studies demonstrated its capacity to efficiently overcome a series of extra- and intracellular barriers in the respiratory tract, such as the glycocalyx, mucus layer, mucociliary clearance, and cell membranes (13, 14, 43). However, in vivo studies demonstrated that rSeV-mediated gene transduction was transient (lasting ∼7 days) and that repeated administration was inefficient (16). The reasons for this transient transduction remain unclear.In contrast, the capacity to efficiently and transiently transduce host cells is of considerable interest from a vaccine vector viewpoint. Indeed, promising rSeV-vectored vaccine candidates have been described for other respiratory viruses, such as respiratory syncytial virus (RSV), hPIV1, hPIV2, hPIV3, and systemic viruses, such as HIV-1 (22, 40, 44). Despite its considerable promise as a viral vector, little is known about how rSeV interacts with human airway epithelial cells (HAE).To address this, we established an ex vivo/in vitro model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs) in air-liquid interface (ALI) cultures. The pediatric origin of the primary bronchial cells allowed us to investigate SeV-host interactions in a pediatric context. The need for CF gene therapy or respiratory virus vaccines for infants or children emphasizes the relevance of this ex vivo/in vitro pediatric model. Using rSeV expressing enhanced GFP (rSeV/eGFP), we comprehensively investigated the consequences of SeV infection in these cultures, including the types of cells infected, virus growth kinetics, cytopathic effects (CPE), and inflammatory responses. Our data provided novel insights into the interaction of SeV with pediatric airway epithelium and the limitations and/or advantages of its use as a vector.     

基于Semliki森林病毒复制子的新型RNAi质粒载体的构建

目的:以semliki森林病毒复制子为基础,构建一类可迅速高效表达shRNA的新型RNAi载体。方法:以Semliki森林病毒衍生的复制子载体pSFV1为骨架,用CMV IE启动子替换SP6启动子并在3′-UTR下游插入SV40 polyA转录终止子,在原26S亚基因组启动子后插入带有相应改良多克隆位点的shRNA表达元件,同时加入抗新霉素选择复合体,并去掉3′-UTR的重复序列。所获载体用于沉默EGFP基因,通过体外细胞转染、病毒颗粒制备、荧光显微镜观察、RT-PCR分析等初步验证、评估其效果。结果:构建了基于Semliki森林病毒复制子的新型RNAi质粒载体pSFV-RNAi Ready。经体外实验初步证实,该载体直接转染细胞,或与辅助载体共转染,制备成具有感染能力的重组病毒颗粒后使用,均可高水平表达shRNA,沉默目的基因。其中使用病毒颗粒抑抑效率可高达90%以上。结论:该载体的成功构建,可望显著拓宽SFV载体的应用范围,丰富RNAi实施手段,并用于相关科学研究及基因药物技术开发。     

Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2–4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.     

Heterokaryon Formation of Simian Virus 40-transformed Cells in the Presence of Ultraviolet-irradiated Sendai Virus

Most simian virus 40-transformed mouse kidney lines form heterokaryons with CV-1 cells in the presence of ultraviolet-irradiated Sendai. However, two nonyielder lines, mKS-U2 and mKS-U20, fuse poorly.     

Derivation of Transgene-Free Human Induced Pluripotent Stem Cells from Human Peripheral T Cells in Defined Culture Conditions

Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.